Cryo-electron microscopy augmented by single particle analysis is an experimental technique that provides information about structure and function of macromolecules. Recent developments in 3D reconstructions of the ribosome in the co-PI's lab have dramatically changed the role that cryo-EM plays in understanding of the translational apparatus, and have provided the first 3D visualizations of tRNA as well as elongation factors in situ on the ribosome. 3D reconstruction of the ribosome in a well- defined conformational state resulted in a density map at 15 Angstrom units resolution. We show in preliminary experiments that further improvements in resolution were possible by increasing the number of particle images to approximately 60,000. In addition, we demonstrate that the conformational variability is the major resolution-limiting factor and we show that a proper choice of resolution measure facilitates selection of homogenous data sets. This proposal describes a 5-years research plan that seeks to develop a unified software framework that will expedite processing of cryo-EM data in excess of 100,000 particle images. To achieve such a throughput, we will design a fully automatic particle picking system that will utilize both textural and a priori structural information about the cryo-EM data. To limit the variability in the data set we will develop particle classification schemes that are based on 3D templates. Mathematical and numerical tools will be implemented that are geared toward a multistage optimization of the structural resolution. Specifically, all the parameters that can be controlled in the process of the structure reconstruction will be identified and linked to the resolution measure used. We expect that by addressing the data collection problem and the problem of conformational heterogenity, we will be able to advance the resolution of single-particle reconstructions substantially. The methods we propose to develop promise to have a high impact in the study of macromolecular structure and interaction.